Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex

Abstract

Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABαC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.

Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

“…Nonetheless, the immunoprecipitation experiments demonstrate that despite the low expression, CLDN1 directly binds to ZO‐1 in HBMECs, and this association becomes more efficient after the miR‐155 downregulation. ZO‐1 levels and localization are influenced by its phosphorylation 8, 34. Therefore, we compared the ZO‐1 phosphorylation state in anti–ZO‐1 immunoprecipitates from OGD/I and OGD/IC cells.…”

Effect of aspirin on the Wnt/β-catenin pathway is mediated via protein phosphatase 2A

“…Briefly, DLD-1 and SW480 cells were preincubated with 100 nM Okadaic Acid (concentration commonly used in cell-based assays (Mistry et al, 1998;Xie et al, 1998), which efficiently inhibits PP2A without affecting protein phosphatase 1 (PP1) (Favre et al, 1997;Nunbhakdi-Craig et al, 2002;Smal et al, 2004)), or with vehicle control. Cells were subsequently incubated with aspirin (5 mM) in the presence of Okadaic acid or vehicle control during a time course (as indicated).…”

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

“…In addition claudin-1 and -4 are substrates for the WNK kinase, although no putative sites have been reported [33]. Some investigations have shown modulation of claudin-4 by aPKC [30] and PKCθ [34]. In Caco-2 cells, constitutively active PKCθ is necessary to maintain barrier function and this process involves the phosphorylation of claudin-1 and -4 [34].…”

“…Interestingly, several studies have demonstrated the involvement of various kinases in the phosphorylation and regulation of claudin proteins [29][30][31][32][33][34][35][36][37], and we have recently shown that phosphorylation of claudin-3 by PKA can affect TJ properties in ovarian cancer cells [38]. Protein kinase C (PKC) isoforms are present in ovarian cancer and are known to modulate TJ function by phosphorylation of the proteins in the complex [24,34,[39][40][41][42][43], but it is unclear whether PKC can directly phosphorylate and regulate claudin proteins.…”